In cultivation of rice, pathogenic microorganisms of plant diseases such as blast have caused problems of decline of the yield and have been controlled mainly by chemicals. The chemical control is generally effective only immediately before the diseases occur but much less effective after the diseases occur, thereby causing serious damages. On the other hand, genetic analyses have shown that various disease-resistant genes might exist on the basis of their chromosome maps (Proteins, Nucleic Acids, Enzymes, vol. 37 (7) 1353 (1992)). It is importantly desirable in rice breeding to introduce the trait of a variety having such a disease-resistant gene, i.e. resistant gene, into cultivated rice plants through the cross-breeding.
Although a number of such resistant genes were presumed to exist in rice plants and were associated with one another on chromosome maps, there were no reports revealing that these genes were isolated and clarified at genetic level.
Some successes have been reported in conferring a resistance to pathogenic microorganisms to plants other than rice, e.g. by introducing both genes of chitinase and .beta.-1,3-glucanase into tobacco plants or the like (M. B. Sela-Buurllage et al., Plant Physiol., 101, 857 (1993)), or introducing a detoxification enzyme gene for toxin produced by tobacco wildfire bacteria into tobacco plants (V. Nikolaeva et al., In vitro, 29A, 3, Pt. 2, 87A (1993)). In maize, a gene for an enzyme which reduces and detoxifies HC toxin (Helminthosporium carbonum toxin) produced by its pathogen Cochliobolus carbonum, i.e. HC toxin reductase, was isolated and proved to be consistent with the resistant locus of the chromosome (G. S. Johal et al., Science, 258, 985 (1992)).
In rice, disease-resistant genes such as genes for chitinase (KIM et al., Biosci. Biotechnol. Biochem. 58 (6) 1164 (1894)), lipoxygenase (JP-A-6-225774), phytoalexin synthetase (Minami et al., Eur. J. Biochem. 185, 19 (1989)), and peroxidase (Ito et al., Plant Cell Report, 13, 361 (1994)) were isolated, but there were no reports evidencing any correlation between these genes and disease resistances. Studies on the resistance mechanism of rice infection with a blast pathogen suggested two mechanisms, one being a mechanism of inhibiting the growth of pathogenic microorganisms by hypersensitive cell death in infected parts of rice plant cells at early infection phase and the other being a mechanism of inhibiting infection by alteration of cell membranes (S. ARASE, Chemistry and Biology, vol. 31 (4), 235 (1993)), but the gene responsible for the disease resistance has not yet been clarified.
Thus, the occurrence of plant diseases, as well as the resistance to plant diseases, has been studied in rice with difficulty and without progress at genetic level, because both the pathogenic infection mechanism and the resistance expression mechanism in plants are very complex and because any correlation could not be obtained even between labo-level resistance and field-level resistance.
However, several disease-resistant genes have been believed to be involved in various resistance mechanisms. Therefore, it will be necessary to isolate a full-length gene resistant to rice diseases including blast; to clarify the structure of the isolated gene as well as the primary structure of the corresponding protein; to express the gene in rice plants; and to examine functions of the gene in detail.
In this context, we have now isolated a gene for NADH-dependent reductase having an ability to detoxify a toxin produced by a pathogen of blast as one of disease-resistance genes, analyzed its structure, and introduced it into plants, thereby finding that the disease resistance could readily be given to plants such as rice. Namely, while keeping in mind the report that the protein expressed from a gene encoding a protein presumed to be the HC toxin reductase isolated from maize as described above offers a resistance by reducing and detoxifying the HC toxin produced by the pathogenic Cochliobolus carbonum, we have now succeeded in isolation of a cDNA clone having a partial sequence highly homologous to the HC toxin reductase gene by the random sequencing of cDNA from a rice anther cDNA library; and in isolation of a full-length rice NADH-dependent reductase cDNA from rice anthers using the obtained cDNA clone as a probe. As a result, it has been evidenced that this gene was associated with the blast-resistant locus.